The present invention is in the general field of treatments of sepsis and other disorders characterized by endothelial cell dysfunction, specifically using thrombin-activable fibrinolysis inhibitor (TAFI) and analogues thereof administered in a therapeutically acceptable amount, alone or in combination with other active compounds such as activated protein C.
Vascular endothelium is comprised of the epithelial cells that form the lining of blood vessels. While vascular endothelium once was thought to be a passive barrier that simply channeled the blood, it now is known that endothelial cells are actively involved in the regulation of intravascular coagulation mechanisms and in the movement of fluid between the parenchyma and the intravascular space. The endothelium of the microvasculature further normally functions as a dynamic semi-permeable membrane. Intracellular mechanisms selectively control the porosity of the cell to various blood components. Normally, the membrane of endothelial cells is permeable to certain small physiologic molecules, such as water and nutrients, and to larger molecules under selected conditions, allowing them to pass as needed to and from the adjacent tissues. However, the endothelium normally is impermeable to larger molecules, such as plasma proteins that must remain in the blood to function. Although the endothelial mechanisms for regulating permeability and preventing thrombosis ordinarily are remarkably efficient, these mechanisms may be disrupted by an inflammatory stimulus which elicits the release of inflammatory mediators, and in particular the monokines tumor necrosis factor (TNF) and interleukin 1 (IL-1). In most cases, the release of these mediators is accompanied by activation of the plasma complement system.
An inflammatory stimulus that leads to release of inflammatory mediators occurs in a wide variety of pathological conditions. These conditions include sepsis, especially gram-negative septic shock, gram positive septic shock of the type caused by Staphylococcus aureus, and injuries involving substantial tissue damage, such as burns and crush injuries. Such a stimulus also may occur in adult respiratory distress syndrome and reperfusion inflammatory syndrome. The endothelial cell surface, as well as that of other cells in contact with the blood (e.g., fixed macrophages), is converted from an anticoagulant to a procoagulant state which permits intravascular coagulation. This, in turn, leads to consumption of coagulation factors, hence the term consumption coagulation. When this dysfunction is systemic, it is referred to as disseminated intravascular coagulopathy (DIC).
The mechanisms for regulating permeability are also affected so that the endothelial cell loses its ability to selectively control porosity. The endothelial cells swell and fluid begins leaking into the surrounding tissues, causing anoxia and parenchymal damage. This is accompanied by increased peripheral resistance, decreased venous return and in many instances, death due to shock. In most instances, for example septic shock, the response of the endothelium to the inflammatory stimuli involves both coagulopathy and abnormal permeability. However, in some conditions, both these dysfunctional responses may not occur. For example, some conditions may involve primarily uncontrolled permeability with minimal or no significant coagulopathy.
Sepsis is an infection-induced syndrome defined as the presence of two or more of the following features of systemic inflammation: fever or hypothermia, leukocytosis or leukopenia, tachycardia, and tachypnea or a supranormal minute ventilation. When an organ system begins to fail because of sepsis, the sepsis is considered severe. Each year, sepsis develops in more than 500,000 patients in the United States, and only 55 to 65 percent of those patients survive.
The major cause for sepsis is believed to be the excessive inflammatory response induced by endotoxin, or lipopolysaccharide (LPS), a component of Gram-negative bacteria. In plasma, LPS is released from the cell wall of growing bacteria, or bacteria damaged by complement or antibiotics. The LPS rapidly forms complexes with a variety of circulating proteins and lipids. Interaction of those free or bound LPS with the host monocytes and macrophages triggers the production of a cascade of proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-alpha), interleukin 1 (IL-1) and interleukin 6 (IL-6). Systemic release of these cytokines with complement activation products and other materials induces excessive inflammatory response in both human and animals during bacteremia or overwhelming Gram-negative infection which may lead to septic shock and death.
More recently, investigations into the time course and extent of coagulation and fibrinolysis abnormalities in sepsis, their relationship to endothelial dysfunction, and the factors that may initiate these changes have highlighted the crucial role of an imbalance in hemostatic mechanisms. This imbalance can manifest as disseminated intravascular coagulopathy (DIC) and microvascular thrombosis, and may ultimately be one of the primary factors driving organ dysfunction and death. The pathophysiology of sepsis is currently viewed as one in which there is an uncontrolled cascade of inflammation, coagulation, and fibrinolysis. At each step in the cycle, auto-amplification processes contribute to the increased acceleration of coagulation abnormalities, inflammation, and endothelial injury.
Early events in the sepsis cascade triggered by the host's immune response have direct damaging actions on the vascular endothelium. Subendothelial structures are exposed and collagenases are liberated. Endothelial cells and monocyte/macrophages express tissue factor (TF), triggering the extrinsic coagulation cascade and accelerating the production of thrombin. Concurrently, the endothelial damage causes further exacerbation of inflammation, resulting in neutrophil activation, neutrophil-endothelial cell adhesion, ischemia reperfusion and further elaboration of inflammatory cytokines. These inflammatory processes further contribute to vascular endothelial dysfunction. Microvascular function is compromised, resulting in decreased tissue perfusion and hypoxemia with resultant organ dysfunction and failure.
Endogenous modulators of homeostasis, such as Protein C and AT-III, are consumed and their levels become deficient as the body attempts to return to a normal functional state. Under normal conditions, the endothelial surface proteins thrombomodulin and endothelial Protein C receptor (EPCR), activate Protein C and its modulating effects. In sepsis, the endothelial damage impairs this function of thrombomodulin and EPCR, thereby contributing to the loss of control. Left unopposed, the endothelial damage accumulates. This uncontrolled cascade of inflammation and coagulation fuels the progression of sepsis, resulting in hypoxia, widespread ischemia, organ dysfunction, and ultimately death for a large number of patients.
Inflammation is the body's normal response to infection. The body's initial response to an infection is to induce a pro-inflammatory state. Pro-inflammatory mediators, such as tumor necrosis factor (TNF-a), interleukin-1 (IL-1), interleukin-6 (IL-6), and platelet-activating factor (PAF) are released. These mediators have multiple overlapping effects designed to repair existing damage and limit new damage. To ensure that the effects of the pro-inflammatory mediators do not become destructive, the body again attempts to maintain its normal functional state by launching compensatory anti-inflammatory mediators, such as interleukin-4 (IL-4) and interleukin-10 (IL-10), which normally downregulate the initial pro-inflammatory response.
In sepsis, regulation of the early response to infection is lost, and a massive systemic reaction occurs. These excessive or inappropriate inflammatory reactions are detrimental. An excess of the inflammatory mediators, such as TNF-alpha, IL-1, and complement activation products (e.g., C5a) are released, triggering an overwhelming physiologic response including neutrophil activation and adherence to the microvascular endothelium resulting in the development of diffuse capillary injury. Finally, excessive inflammatory reactions interfere with normal tissue function, leading to tissue damage and organ dysfunction.
In sepsis, the processes of inflammation and coagulation are intimately linked. Multiple inflammatory mediators that are released to fight infection also promote coagulation, which contributes to sepsis. In addition, the infectious agent itself can cause endothelial damage, which also promotes coagulation. Coagulation factors are activated when blood comes into contact with sub-endothelial connective tissues or with negatively charged surfaces that are exposed as a result of tissue damage. The first step is the binding of factor XII to a sub-endothelial surface exposed by an injury, thereby activating Factor XII. The activated factor XII activates factor XI. Eventually factor X is activated by a complex of molecules containing activated factor IX, factor VIII, calcium, and phospholipid. The end result of the clotting pathway is the production of thrombin, which converts soluble fibrinogen to fibrin. The insoluble fibrin aggregates and forms a clot, together with aggregated platelets (thrombi), blocking the damaged blood vessel and preventing further bleeding. In sepsis, multiple pro-inflammatory cytokines, such as IL-1alpha, IL-1beta, and TNF-alpha, induce the expression of TF on endothelial cells and monocytes, initiating coagulation. Tissue factor is a key mediator between the immune system and coagulation, and is the principal activator of coagulation. Tissue factor interacts with factor VIIa, forming the factor VIIa-TF complex, which activates factors X and IX. Amplification of coagulation via thrombin-mediated processes occurs with activated factors XI, VIII, and V. In the final stage, large amounts of thrombin are generated. Fibrin threads form a clump with activated platelets at the site of endothelial damage and a stable clot is formed.
Numerous investigational compounds have been studied for the treatment of sepsis. Many of these investigational therapies may have been unsuccessful because they modulate only a single pathophysiologic component of sepsis. Therapies targeting a wider range of mechanisms may be required to treat a disease process as complex as sepsis. Recently, the use of activated Protein C (“APC”) to treat sepsis has been tested in phase III clinical trials by Eli Lilly. The APC was shown to be effective in reducing all cause mortality of 30% by 19%. However, approximately 24% of patients still die and it is desirable to have additional or alternative treatments for sepsis and other means of treating disorders characterized by endothelial cell dysfunction.
It is therefore an object of the present invention to provide a method and pharmaceutical compositions for treatment of sepsis and other disorders characterized by dysfunctional endothelium.